Enhanced ligation reactions

ABSTRACT

In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of at least one agent that generates a ligatable terminal 5′ phosphate group by removing an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, an aprataxin enzyme can catalyze removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of an aprataxin enzyme under conditions suitable for ligating nucleic acid ends.

This subject application is a Continuation Application of U.S.application Ser. No. 15/695,244, filed Sep. 5, 2017, which is aContinuation Application of U.S. application Ser. No. 15/071,086, filedMar. 15, 2016, now U.S. Pat. No. 9,765,388, which is a ContinuationApplication of U.S. application Ser. No. 14/360,892, filed May 27, 2014,now U.S. Pat. No. 9,315,861, which is a U.S. National Application filedunder 35 U.S.C. § 371 of International Application No.PCT/US2012/066869, filed on Nov. 28, 2012, which claims the benefit ofpriority under 35 U.S.C. § 119 to U.S. Provisional Application No.61/564,243, filed on Nov. 28, 2011; and U.S. application Ser. No.14/360,892 also claims priority to U.S. application Ser. No. 13/980,280,filed on Jul. 17, 2013, which is a U.S. National Application filed under35 U.S.C. § 371 of International Application No. PCT/US2012/021465,filed Jan. 17, 2012, which claims the benefit of priority under 35U.S.C. § 119 to U.S. Provisional Application Nos. U.S. 61/433,488, filedon Jan. 17, 2011, U.S. 61/433,502, filed on Jan. 17, 2011, U.S.61/474,168, filed on Apr. 11, 2011, and U.S. 61/474,205, filed on Apr.11, 2011, the disclosures each of which are incorporated herein byreference in their entireties.

Throughout this application various publications, patents, and/or patentapplications are referenced. The disclosures of these publications,patents, and/or patent applications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The instant application contains a Sequence Listing which is submittedherewith in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 14, 2012, isnamed LT00621PCT_Sequence_Listing_ST25.txt, and is 20 kilobytes in size.

FIELD

Enhancement of nucleic acid ligation efficiency comprises an enzyme thatresolves abortive ligation intermediates.

INTRODUCTION

DNA ligases join together nucleic acid ends by forming a phosphodiesterbond at single-stranded or double-stranded breaks in a DNA duplex.Generally, a mechanism for DNA ligases involves production of anadenylated intermediate that can fail to undergo formation of aphosphodiester bond to join the ends. Production of these adenylatedintermediates can reduce ligation efficiency.

SUMMARY

In some embodiments, the present teachings provide compositions,systems, methods and kits for ligation of two nucleic acid ends. Nucleicacid ligation includes joining together two nucleic acid ends. Methodsfor nucleic acid ligation comprise two or more enzymes. In someembodiments, a ligation reaction can include two or more enzymaticreactions. In some embodiments, an enzymatic reaction includes forming aphosphodiester bond between two nucleic acid ends. A reaction catalyzedby a first enzyme can sometimes produce intermediates that fail to join.In some embodiments, a second enzymatic reaction includes resolving thefailed intermediates to produce termini that undergo successfulligation.

In some embodiments, the present teachings provide a method for nucleicacid ligation comprising: conducting a nucleic acid ligation reaction inthe presence of one or more agents that catalyze removal of an adenylategroup from a terminal 5′ phosphate of a nucleic acid.

Optionally, methods for nucleic acid ligation comprise: conducting anucleic acid ligation reaction in the presence of at least one agentthat generates a ligatable terminal 5′ phosphate group by removing anadenylate group from a terminal 5′ phosphate of a nucleic acid.

Optionally, methods for nucleic acid ligation comprise: conducting anucleic acid ligation reaction in the presence of an aprataxin enzymeunder conditions suitable for ligating nucleic acids together.

Optionally, methods for nucleic acid ligation comprise a ligase enzyme.

Optionally, methods for nucleic acid ligation comprise two nucleic acidends.

Optionally, methods for nucleic acid ligation comprise: contacting twonucleic acid ends with at least one ligase enzyme and at least oneaprataxin enzyme under conditions suitable for ligating nucleic acidends

Optionally, methods for nucleic acid ligation comprise: contacting anadenylated nucleic acid with an aprataxin enzyme under conditionssuitable for ligating nucleic acid ends and suitable for aprataxinactivity.

Optionally, methods for nucleic acid ligation comprise: closing asingle-stranded nick on a nucleic acid duplex. In some embodiments, thenick is formed by a first and second oligonucleotide hybridized to apolynucleotide, wherein the first and second oligonucleotides abut eachother.

Optionally, the nucleic acid ligation reaction comprises: joiningtogether two ends of two nucleic acids. In some embodiments, at leastone end of one of the nucleic acids is attached to a surface. In someembodiments, the surface comprises a planar surface, bead or particle.

Optionally, the nucleic acid ligation reaction comprises: joiningtogether two ends of a single nucleic acid to circularize the singlenucleic acid.

In some embodiments, the present teachings provide a method for nucleicacid ligation comprising: conducting at least one cycle of a repetitivecycle ligation reaction in the presence of an aprataxin enzyme underconditions suitable for ligating nucleic acid ends. In some embodiments,the repetitive cycle ligation reaction comprises: a ligase chainreaction (LCR), gap LCR, or ligation detection reaction (LDR).

Optionally, the aprataxin enzyme comprises an amino acid sequenceaccording to SEQ ID NO: 1.

Optionally, the ligase comprises a mesophilic or thermostable ligaseenzyme. In some embodiments, the ligase comprises an E. coli DNA ligase,Taq DNA ligase, 9° N DNA ligase, or T4 DNA ligase.

Optionally, the ligase comprises a small footprint ligase enzyme. Insome embodiments, the small footprint ligase joins together apolynucleotide to an oligonucleotide comprising 8, 7, 6, 5, 4, 3 or 2nucleotides in length. In some embodiments, the small footprint ligasecomprises an amino acid sequence of any one of SEQ ID NOS:3-8.

In some embodiments, the present teachings provide a method for joiningtogether a plurality of unbound nucleic acids to a plurality ofimmobilized nucleic acids, comprising: contacting the plurality ofunbound nucleic acids and the plurality of immobilized nucleic acidswith at least one ligase enzyme and at least one aprataxin enzyme underconditions suitable for nucleic acid ligation. In some embodiments, oneend of the plurality of immobilized nucleic acids is attached to asurface. In some embodiments, the surface comprises a planar surface, abead or a particle. In some embodiments, the plurality of immobilizednucleic acids joined to the plurality of unbound nucleic acids issubjected to a sequencing reaction.

In some embodiments, the present teachings provide a method forpreparing a nucleic acid-templated surface, comprising: contacting (i) aplurality of nucleic acids that are attached to a surface (e.g.,immobilized) and (ii) a plurality of unbound nucleic acids with at leastone ligase enzyme and at least one aprataxin enzyme under conditionssuitable for nucleic acid ligation thereby generating a plurality oftemplated nucleic acids. In some embodiments, the method furthercomprises sequencing the plurality of templated nucleic acids. In someembodiments, the plurality of immobilized nucleic acids joined to theplurality of unbound nucleic acids is subjected to a sequencingreaction.

In some embodiments, the present teachings provide a method forsequencing comprising: (a) hybridizing a template polynucleotide to afirst and second oligonucleotide probe so that the first and secondoligonucleotide probes abut each other to form a nick; (b) contactingthe nick with at least one aprataxin enzyme and at least one ligaseenzyme to close the nick; and (c) detecting the distinctive detectablereporter moiety so as to determine the sequence of the first or secondoligonucleotide probe hybridized to the template polynucleotide.Optionally, the first or second oligonucleotide probe can be labeledwith a distinctive detectable reporter moiety. Optionally, the templatepolynucleotide can be attached to a surface. Optionally, the aprataxinenzyme comprises an amino acid sequence according to SEQ ID NO:1.Optionally, the ligase comprises a small footprint ligase having anamino acid sequence according to any one of SEQ ID NOS:3-8.

In some embodiments, the present teaching provide a method forsequencing comprising: (a) hybridizing a template polynucleotide to afirst and second oligonucleotide probe so that the first and secondoligonucleotide probes abut each other to form a first nick, wherein thesecond oligonucleotide probe is labeled with a first distinctivedetectable reporter moiety; (b) contacting the first nick with at leastone aprataxin enzyme and at least one ligase enzyme to close the firstnick (e.g., join together the first and second oligonucleotide probes);(c) detecting the first distinctive detectable reporter moiety therebydetermining the sequence of the second oligonucleotide probe hybridizedto the template polynucleotide; (d) hybridizing the templatepolynucleotide to a third oligonucleotide probe so that the third andsecond oligonucleotide probes abut each other to form a second nick,wherein the third oligonucleotide probe is labeled with a seconddistinctive detectable reporter moiety; (e) contacting the second nickwith at least one aprataxin enzyme and at least one ligase enzyme toclose the second nick; and (f) detecting the second distinctivedetectable reporter moiety thereby determining the sequence of the thirdoligonucleotide probe hybridized to the template polynucleotide.Optionally, the template polynucleotide can be attached to a surface.

In some embodiments, the present teaching provide a method forsequencing comprising: (a) hybridizing a template polynucleotide to afirst and second oligonucleotide probe so that the first and secondoligonucleotide probes abut each other to form a first nick, wherein thesecond oligonucleotide probe is labeled with a first distinctivedetectable reporter moiety; (b) contacting the first nick with at leastone aprataxin enzyme and at least one ligase enzyme to close the firstnick (e.g., join together the first and second oligonucleotide probes);(c) detecting the first distinctive detectable reporter moiety therebydetermining the sequence of the second oligonucleotide probe hybridizedto the template polynucleotide; (d) hybridizing the templatepolynucleotide to a third and fourth oligonucleotide probe so that thethird and fourth oligonucleotide probes abut each other to form a secondnick, wherein the fourth oligonucleotide probe is labeled with a seconddistinctive detectable reporter moiety; (e) contacting the second nickwith at least one aprataxin enzyme and at least one ligase enzyme toclose the second nick; and (f) detecting the second distinctivedetectable reporter moiety thereby determining the sequence of thefourth oligonucleotide probe hybridized to the template polynucleotide.Optionally, the template polynucleotide can be attached to a surface.

In some embodiments, the present teachings provide a compositioncomprising two nucleic acid ends ligated together according to themethods for nucleic acid ligation disclosed herein.

In some embodiments, the present teachings provide a compositioncomprising a first and second oligonucleotide hybridized to apolynucleotide and ligated together according to the methods for nucleicacid ligation disclosed herein.

In some embodiments, the present teachings provide a compositioncomprising two ends of two nucleic acids ligated together according toaccording to the methods for nucleic acid ligation disclosed herein.

In some embodiments, the present teachings provide a compositioncomprising two ends of a single nucleic acid joined together accordingto the methods for nucleic acid ligation disclosed herein.

In some embodiments, the present teachings provide a compositioncomprising a product of at least one cycle of a repetitive cycleligation reaction generated according to the methods for nucleic acidligation disclosed herein.

In some embodiments, the present teachings provide a compositioncomprising a plurality of unbound nucleic acids ligated to a pluralityof immobilized nucleic acids according to the methods for nucleic acidligation disclosed herein.

In some embodiments, the present teachings provide an isolated aprataxinenzyme comprises an amino acid sequence according to SEQ ID NO:1.

In some embodiments, the present teachings provide a small footprintligase comprises an amino acid sequence of any one of SEQ ID NOS:3-8.

DRAWINGS

FIG. 1 is a schematic depicting non-limiting embodiments of a generalmechanism for DNA ligation.

FIG. 2A is a non-limiting embodiment of an amino acid sequence of ahuman aprataxin enzyme (SEQ ID NO:1).

FIG. 2B is a non-limiting embodiment of a nucleotide sequence of a humanaprataxin enzyme (SEQ ID NO:2).

FIG. 3 is a non-limiting embodiment of an amino acid sequence of aligase from Paramecium bursaria Chlorella virus (SEQ ID NO:3).

FIG. 4 is a non-limiting embodiment of an amino acid sequence of aligase from Burkholderia pseudomallei (SEQ ID NO:4).

FIG. 5 is a non-limiting embodiment of an amino acid sequence of aligase from Haemophilus influenza (SEQ ID NO:5).

FIG. 6 is a non-limiting embodiment of an amino acid sequence of anartificial ligase derived from Haemophilus influenza (SEQ ID NO:6).

FIG. 7 is a non-limiting embodiment of an amino acid sequence of anartificial ligase derived from Haemophilus influenza (SEQ ID NO:7).

FIG. 8 is a non-limiting embodiment of an amino acid sequence of aligase from Haemophilus influenza (SEQ ID NO:8).

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control. Unless defined otherwise, all technicaland scientific terms used herein have the same meaning as is commonlyunderstood by one of ordinary skill in the art to which these inventionsbelong. All patents, patent applications, published applications,treatises and other publications referred to herein, both supra andinfra, are incorporated by reference in their entirety. If a definitionand/or description is set forth herein that is contrary to or otherwiseinconsistent with any definition set forth in the patents, patentapplications, published applications, and other publications that areherein incorporated by reference, the definition and/or description setforth herein prevails over the definition that is incorporated byreference. It will be appreciated that there is an implied “about” priorto the temperatures, concentrations, times, etc discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein. In this application, the use ofthe singular includes the plural unless specifically stated otherwise.Also, the use of “comprise”, “comprises”, “comprising”, “contain”,“contains”, “containing”, “include”, “includes”, and “including” are notintended to be limiting. As used herein, the terms “comprises,”“comprising,” “includes,” “including,” “has,” “having” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a process, method, article, or apparatus that comprises a listof features is not necessarily limited only to those features but mayinclude other features not expressly listed or inherent to such process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive-or and not to an exclusive-or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent). It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present teachings described herein shall have themeanings that are commonly understood by those of ordinary skill in theart. Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are used,for example, for nucleic acid purification and preparation, chemicalanalysis, recombinant nucleic acid, and oligonucleotide synthesis.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications or as commonly accomplished in the artor as described herein. The techniques and procedures described hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the instant specification. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Thirded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.2000). The nomenclatures utilized in connection with, and the laboratoryprocedures and techniques described herein are those well known andcommonly used in the art.

As utilized in accordance with exemplary embodiments provided herein,the following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein the terms “ligate” and “ligation” refer to joiningtogether two free nucleic acid ends by forming a phosphodiester bondbetween the two free ends. In some embodiments, ligation can includejoining together two ends of two nucleic acid strands or joiningtogether two ends of a single nucleic acid strand (e.g.,circularization). Ligation can include joining together two ends of twosingle-stranded nucleic acids or joining together two ends twodouble-stranded nucleic acids. Ligation can include closing asingle-stranded nick in a nucleic acid duplex.

As used herein the term “nick” refers to a location on a double-strandednucleic acid that lacks a phosphodiester bond between adjacentnucleotides of one of the nucleic acid strands, while the other strandhas adjacent nucleotides joined by a phosphodiester bond at that samelocation. In some embodiments, a phosphodiester bond includes analoglinkages that join adjacent nucleotides (or nucleotide analogs).

As used herein the term “adenylated nucleic acid” and “adenylated DNA”refers to an adenylate group that is covalently linked to a nucleicacid. For example, an “adenylated nucleic acid” comprises an adenylategroup that is covalently linked to a terminal 5′-phosphate of a nucleicacid or an adenosine-5′-phosphate linked to a terminal 5′-phosphate of anucleic acid.

As used herein the terms “nucleic acids”, “oligonucleotides” and“polynucleotides” refers to single-stranded or double-stranded nucleicacids, and includes DNA, RNA, chimeric RNA/DNA, and derivatives thereof.

DESCRIPTION OF VARIOUS EMBODIMENTS

In some embodiments, the present teachings provide compositions,systems, methods and kits for enhanced ligation of two nucleic acidends.

A general mechanism for DNA ligation involves several steps, includingbut not limited to: (1) a ligase enzyme reacts with ATP to form anenzyme-adenylate complex (with an activated AMP) and releases ADP; (2)the enzyme-adenylate complex transfers the activated AMP to a5′-terminal phosphate of a nucleic acid to form an adenylated DNAstrand; and (3) the enzyme catalyzes formation of a phosphodiester bondbetween the adenylated DNA and a terminal 3′OH end of a nucleic acid tojoin together the two nucleic acid ends and release of AMP (FIG. 1).

An abortive ligation reaction can result from formation of adenylatedDNA intermediates and failure to proceed to formation of phosphodiesterbonds. Abortive ligation reactions can reduce ligation reactionefficiency. Aborted ligation reactions can be rescued by reversing theadenylation step to restore DNA molecules having a terminal 5′ phosphategroup which can proceed to adenylation and phosphodiester bondformation.

Aprataxin enzyme can reverse the DNA adenylation reaction to produce anucleic acid having a ligatable end. Aprataxin can resolve aborted DNAligation intermediates by catalyzing nucleophilic release of adenylategroups that are linked to a terminal 5′-phosphate group to regenerate aterminal 5′-phosphate group that can be joined via phosphodiester bondformation (Ahel 2006 Nature 443:713-716).

In some embodiments, compositions, systems, methods and kits forenhancing ligation of nucleic acids comprise ligase and aprataxinenzymes. Aprataxin can enhance a ligation reaction by improving theefficiency of a nucleic acid ligation reaction. Aprataxin can improvethe number-fold and/or rate of nucleic acid ligation by resolvingaborted adenylated ligation intermediates.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise joining together two nucleic acid ends. In someembodiments, methods for enhancing a nucleic acid ligation reactioncomprise closing a single-stranded nick on a nucleic acid duplex orjoining together two ends of two nucleic acids or joining together twoends of a single nucleic acid (e.g., circularization).

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: conducting a nucleic acid ligation reaction in thepresence of one or more agents that catalyze removal of an adenylategroup from a terminal 5′ phosphate of a nucleic acid. In someembodiments, methods for enhancing a nucleic acid ligation reactioncomprise: conducting a nucleic acid ligation reaction in the presence ofat least one agent that generates a ligatable terminal 5′ phosphategroup by removing an adenylate group from a terminal 5′ phosphate of anucleic acid. In some embodiments, an aprataxin enzyme can catalyzeremoval of an adenylate group from a terminal 5′ phosphate of a nucleicacid.

In some embodiments, methods for joining together two nucleic acid endscomprises: conducting a nucleic acid ligation reaction in the presenceof one or more agents that catalyze removal of an adenylate group from aterminal 5′ phosphate of a nucleic acid. In some embodiments, methodsfor joining together two nucleic acid ends comprises: conducting anucleic acid ligation reaction in the presence of at least one agentthat generates a ligatable terminal 5′ phosphate group by removing anadenylate group from a terminal 5′ phosphate of a nucleic acid. In someembodiments, an aprataxin enzyme can catalyze removal of an adenylategroup from a terminal 5′ phosphate of a nucleic acid.

In some embodiments, a ligation reaction comprises forming aphosphodiester bond between the termini of two nucleic acid ends. Insome embodiments, a ligation reaction comprises forming a phosphodiesterbond between a 5′ terminus of a first nucleic acid end and a 3′ terminusof a second nucleic acid end.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: conducting a nucleic acid ligation reaction in thepresence of an aprataxin enzyme under conditions suitable for ligatingnucleic acid ends.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: contacting two nucleic acid ends with at least oneligase enzyme and at least one aprataxin enzyme under conditionssuitable for ligating nucleic acid ends.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: conducting at least one cycle of a repetitive cycleligation reaction in the presence of an aprataxin enzyme underconditions suitable for ligating nucleic acid ends.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: contacting a ligase enzyme and an aprataxin enzymewith (i) a single-stranded nick on a nucleic acid duplex or (ii) twodouble-stranded nucleic acids or (iii) two single-stranded nucleicacids, under conditions suitable for ligating nucleic acid ends.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: contacting an adenylated nucleic acid with anaprataxin enzyme under conditions suitable for ligating nucleic acidends (e.g., ligation reaction) and suitable for aprataxin activity.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: contacting in any order and in any combination anadenylated nucleic acid, a ligase enzyme and an aprataxin enzyme, underconditions suitable for ligating nucleic acid ends and suitable foraprataxin activity.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: contacting an adenylated nucleic acid simultaneously(e.g., essentially simultaneously) or sequentially with a ligase enzymeand an aprataxin enzyme.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise: contacting two nucleic acid ends with a ligase enzymeand an aprataxin enzyme in the same or different reaction vessels.

In some embodiments, a ligation reaction comprises joining togethernucleic acid ends, where at least one end of a nucleic acid can beattached to a surface (e.g., immobilized). For example, a nucleic acidend that will not be joined to another nucleic acid end can be attachedto a surface.

In some embodiments, methods for enhancing a nucleic acid ligationreaction include conducting a nucleic acid ligation reaction in thepresence of an aprataxin enzyme to increase the number of ligatednucleic acid products compared to the number of ligated nucleic acidproducts resulting from a ligation reaction lacking an aprataxin enzyme.For example, an increase in the number of ligated nucleic acid productscan range from about 1-5%, or about 10-20%, or about 20-30%, or about30-40%, or about 40-50%, or a higher percentage increase in formation ofligation products.

In some embodiments, a suitable nucleic acid ligation condition includeswell known parameters, such as: time, temperature, pH, buffers,reagents, cations, salts, co-factors, nucleotides, nucleic acids, andenzymes. In some embodiments, a nucleic acid ligation reaction can beconducted with a reagent that includes ATP and/or NAD. In someembodiments, a reagent or buffer can include a source of ions, such asKCl, K-acetate, NH₄-acetate, K-glutamate, NH₄Cl, or ammonium sulfate. Insome embodiments, a reagent or buffer can include a source of ions, suchas magnesium, manganese, cobalt, or calcium. In some embodiments, areagent or buffer can include acetate or chloride. In some embodiments,a buffer can include Tris, Tricine, HEPES, MOPS, ACES, MES, or inorganicbuffers such as phosphate or acetate-based buffers which can provide apH range of about 4-12. In some embodiments, a buffer can includechelating agents such as EDTA or EGTA. In some embodiments, a buffer caninclude dithiothreitol (DTT), glycerol, spermidine, BSA (bovine serumalbumin) and/or Tween.

In some embodiments, a suitable condition includes conducting a nucleicacid ligation reaction for a time, such as about 1-10 seconds, or about10-60 seconds, or about 1-30 minutes, or about 30-60 minutes, or about1-3 hours, or about 3-6 hours, or about 6-12 hours, or about 12-24hours, or longer.

In some embodiments, a suitable condition includes conducting a nucleicacid ligation reaction under thermo-cycle conditions, or isothermaltemperature conditions, or a combination of both. In some embodiments, asuitable condition includes conducting a nucleic acid ligation reactionat a temperature range of about 0-10° C., or about 10-20° C., or about20-30° C., or about 30-40° C., or about 40-50° C., or about 50-60° C.,or about 60-70° C., or about 70-80° C., or about 80-90° C., or about90-99° C., or a higher temperature range.

In some embodiments, a suitable condition includes conducting a nucleicacid ligation reaction at a pH range of about 5-9, or a pH range ofabout 6-8, or a pH range of about 7-7.5.

In some embodiments, a suitable condition includes conducting a nucleicacid ligation reaction in a tube, well or flowcell. In some embodiments,the well can be a part of an array or a multi-well plate or a multi-wellchip.

In some embodiments, a ligation reaction comprises closing asingle-stranded nick on a nucleic acid duplex. For example, a first andsecond oligonucleotide can be hybridized to a polynucleotide. The firstand second oligonucleotides can abut each other, while hybridized to thepolynucleotide, to create a nick. In some embodiments, methods forclosing a nick comprise contacting two nucleic acid ends that form thenick with a ligase and aprataxin enzyme.

In some embodiments, a first or second oligonucleotide or thepolynucleotide can be labeled with a detectable reporter moiety (e.g., afluorophore, luminophore, chemiluminophore, or bioluminophore). In someembodiments, a first or second oligonucleotide or the polynucleotide canbe attached to a solid surface. In some embodiments, a first or secondoligonucleotide or the polynucleotide can include an internal orterminal scissile linkage such as a phosphoramidate, phosphorothioate,or phosphorodithiolate linkage.

In some embodiments, the polynucleotide can comprise a single-strandednucleic acid template, and the first and second oligonucleotides cancomprise labeled or non-labeled oligonucleotide probes. In someembodiments, the first or second oligonucleotide probes comprise 4-500nucleotides in length or longer. In some embodiments, the first andsecond oligonucleotide probes abut each other while hybridized to thepolynucleotide template. In some embodiments, the first and secondoligonucleotide probes comprise nucleotide sequences that arecomplementary to at least a portion of the polynucleotide template. Insome embodiments, hybridization of the first and second oligonucleotideprobes to the polynucleotide template forms a nick. In some embodiments,closing the nick by ligation with a ligase in the presence of anaprataxin can increase ligation efficiency. In some embodiments,hybridizing first and second oligonucleotide probes to a polynucleotidetemplate to form a nick, and closing the nick with a ligase reaction inthe presence of an aprataxin enzyme can be used for determining thesequence of the polynucleotide template. In some embodiments, thehybridization and/or closing-the-nick steps can be conducted underisothermal or thermo-cyclic conditions. In some embodiments, the ligaseand/or aprataxin can be a mesophilic or thermostable enzyme. In someembodiments, aprataxin can be used to enhance ligation reactions asconducted in a SOLiD sequencing reaction (WO 2006/084132) by AppliedBiosystems (now part of Life Technologies, Carlsbad, Calif.).

In some embodiments, a method for sequencing comprises: (a) hybridizinga template polynucleotide to a first and second oligonucleotide probe sothat the first and second oligonucleotide probes abut each other whenhybridized to the polynucleotide to form a nick, wherein the first orsecond oligonucleotide probe is labeled with a distinctive detectablereporter moiety; (b) contacting the nick with at least one aprataxinenzyme and at least one ligase enzyme to close the nick (e.g., jointogether the first and second oligonucleotide probes); and (c) detectingthe distinctive detectable reporter moiety so as to determine thesequence of the first or second oligonucleotide probe hybridized to thetemplate polynucleotide. In some embodiments, steps (a)-(c) can berepeated, for example by hybridizing the template polynucleotide to athird oligonucleotide probe so that the third and second oligonucleotideprobes abut each other when hybridized to the polynucleotide to form asecond nick, wherein the third oligonucleotide probe is labeled with asecond distinctive detectable reporter moiety; (b) contacting the secondnick with at least one aprataxin enzyme and at least one ligase enzymeto close the second nick; and (c) detecting the second distinctivedetectable reporter moiety so as to determine the sequence of the thirdoligonucleotide probe hybridized to the template polynucleotide. In someembodiments, first, second and third oligonucleotide probes can havedifferent sequences. In some embodiments, first, second and thirdoligonucleotide probes can be labeled with different or distinctdetectable reporter moieties to distinguish the different sequences ofthe probes. In some embodiments, methods for sequencing comprise a SOLiDsequencing reaction (WO 2006/084132). In some embodiments, steps (a)-(c)can be repeated, for example by hybridizing the template polynucleotideto a third and forth oligonucleotide probe so that the third and fourtholigonucleotide probes abut each other when hybridized to thepolynucleotide to form a second nick, wherein the third or fourtholigonucleotide probe is labeled with a second distinctive detectablereporter moiety; (b) contacting the second nick with at least oneaprataxin enzyme and at least one ligase enzyme to close the secondnick; and (c) detecting the second distinctive detectable reportermoiety so as to determine the sequence of the third or fourtholigonucleotide probe hybridized to the template polynucleotide. In someembodiments, first, second, third and fourth oligonucleotide probes canhave different sequences. In some embodiments, first, second, third andfourth oligonucleotide probes can be labeled with different or distinctdetectable reporter moieties to distinguish the different sequences ofthe probes. In some embodiments, methods for sequencing comprise a SOLiDsequencing reaction (WO 2006/084132).

In some embodiments, methods for closing a nick comprise: contacting anick with a small footprint ligase and an aprataxin enzyme underconditions suitable for ligase activity. Non-limiting examples of smallfootprint ligases are shown in FIGS. 3-8 (SEQ ID NOS: 3-8). Anon-limiting example of aprataxin is shown in FIG. 2A (SEQ ID NO: 1).

In some embodiments, a ligation reaction comprises joining together twoends of two nucleic acids with a ligase and aprataxin enzyme. In someembodiments, the two nucleic acids comprise single- or double-strandednucleic acids. In some embodiments, a ligation reaction comprisesjoining together two blunt ends or two overhang ends. For example, aligation reaction comprises joining together a first end of a firstnucleic acid with a first end of a second nucleic acid. In someembodiments, a first and/or second nucleic acid can be any combinationof a nucleic acid adaptor, polynucleotide-of-interest, target sequence,template, insert sequence, fragment library construct and/or mate pairlibrary construct. In some embodiments, a first or second nucleic acidcan be unbound (non-immobilized) or can be immobilized.

In some embodiments, a ligation reaction comprises joining together aplurality of unbound nucleic acids to a plurality of immobilized nucleicacids with a ligase and aprataxin enzyme. In some embodiments, joiningtogether a plurality of unbound nucleic acids to a plurality ofimmobilized nucleic acids comprises contacting the plurality of unboundnucleic acids and the plurality of immobilized nucleic acids with atleast one ligase enzyme and at least one aprataxin enzyme.

In some embodiments, the plurality of unbound nucleic acids compriseun-amplified nucleic acids, or comprise nucleic acids resulting from anamplification reaction. In some embodiments, a linear amplification orexponent amplification reaction can generate a plurality of unboundnucleic acids. In some embodiments, a PCR or emPCR reaction can generatea plurality of unbound nucleic acids.

In some embodiments, a plurality of unbound nucleic acids can be in anaqueous solution, or can be in an aqueous compartment of an oilemulsion. In some embodiments, immobilized nucleic acids can be in anaqueous solution, or can be in an aqueous compartment of an oilemulsion. In some embodiments, the plurality of unbound nucleic acidsand the immobilized nucleic acids reside in the same aqueous solution orthe same aqueous compartment of an oil emulsion during amplification ofthe unbound nucleic acids. In some embodiments, the plurality of unboundnucleic acids and the immobilized nucleic acids reside in separateaqueous solutions or separate aqueous compartments of an oil emulsionduring amplification of the unbound nucleic acids.

In some embodiments, joining together the plurality of unbound nucleicacids to the plurality of immobilized nucleic acids with a ligase andaprataxin enzyme can be conducted under isothermal or thermo-cyclicconditions. In some embodiments, the ligase and/or aprataxin can be amesophilic or thermostable enzyme.

In some embodiments, the immobilized nucleic acids comprise nucleicacids attached to a surface (e.g., planar, bead or particle). In someembodiments, a plurality of unbound nucleic acids comprises a pluralityof template nucleic acids. In some embodiments, joining together aplurality of unbound nucleic acids (e.g., templates) to a plurality ofimmobilized nucleic acids with a ligase and aprataxin enzyme generatesone or more nucleic acid-templated surfaces (e.g., nucleic acidsattached to a surface). In some embodiments, methods for preparing anucleic acid-templated surface comprise: contacting at least one ligaseenzyme and at least one aprataxin enzyme with (i) a plurality of nucleicacids that are attached to a surface and (ii) a plurality of unboundnucleic acids under conditions suitable for nucleic acid ligation. Insome embodiments, nucleic acid-templated surfaces can be subjected toany next generation sequencing reaction. For example, sequencingreagents can be delivered to the nucleic acid-templated surfaces toconduct a sequencing reaction. In some embodiments, a sequencingreaction includes any next generation sequencing reaction, including:sequencing by oligonucleotide probe ligation and detection (e.g., SOLiD™from Life Technologies, WO 2006/084131); probe-anchor ligationsequencing (e.g., Complete Genomics™ or Polonator™);sequencing-by-synthesis (e.g., Genetic Analyzer, HiSeq™ and MiSeq™ fromIllumina); pyrophosphate sequencing (e.g., Genome Sequencer FLX from 454Life Sciences); ion-sensitive sequencing (e.g., Personal Genome Machinefrom Ion Torrent™ Systems, Life Technologies); and single moleculesequencing platforms (e.g., HeliScope™ from Helicos). In someembodiments, a sequencing reaction comprises use of a chain-terminatingnucleotide, including reversible chain-terminating nucleotides (U.S.Pat. No. 7,476,734, Liu; U.S. Pat. No. 7,713,698, Ju; U.S. Pat. No.8,158,346; Balasubramanian; U.S. Pat. No. 7,771,973, Milton; U.S. Pat.No. 8,088,575, Ju; U.S. Pat. No. 7,414,116, Milton; U.S. Pat. No.7,785,796, Balasubramanian; U.S. Pat. No. 7,345,159, Ju; U.S. Pat. No.7,816,503, Milton; U.S. Pat. No. 7,713,689, Ju; U.S. Pat. No. 7,883,869,Ju; U.S. Pat. No. 7,541,444, Milton; U.S. Pat. Nos. 8,148,064 and8,158,346, Balasubramanian; U.S. Pat. Nos. 7,713,698 and 7,790,869, Ju;U.S. Pat. No. 8,298,792, Ju; U.S. Pat. No. 7,771,973, Milton; U.S. Pat.No. 6,664,079, Ju; and U.S. Pat. No. 7,635,578, Ju).

In some embodiments, a ligation reaction comprises joining together twoends of a single nucleic acid. In some embodiments, a ligation reactioncomprises a ligase and aprataxin enzyme to improve the number-foldand/or rate of nucleic acid ligation. In some embodiments, the singlenucleic acid comprises a single- or double-stranded nucleic acid. Insome embodiments, a ligation reaction comprises joining together twoblunt ends or two overhang ends. For example, a ligation reactioncomprises joining together a first and second end of a first nucleicacid.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise conducting at least one cycle of a repetitive cycleligation reaction with a ligase enzyme and an aprataxin enzyme. In someembodiments, the ligase enzyme can be a mesophilic or thermostableligase enzyme.

In some embodiment, a repetitive cycle ligation reaction generallycomprises three steps: (a) probe hybridization; (b) ligation; and (c)detection. In some embodiments, steps (a)-(c) can be repeated at leastonce. In some embodiments, a probe hybridization step compriseshybridizing a pair of oligonucleotide probes to a template/targetpolynucleotide such that the probes hybridize in close proximity to each(e.g., to form a nick or gap). In some embodiments, a ligation stepcomprises joining together a pair of hybridized oligonucleotide probesto generate ligated probe products. In some embodiments, ligation may bedisrupted if the hybridized probes lack complementarity to thetemplate/target polynucleotide. For example, probes having partialcomplementarity to the template/target polynucleotide may not be ligatedtogether. In some embodiments, the detecting step comprises detectingthe presence of the ligated probe products. In some embodiments,amplification of the ligated probe products can occur prior to, orsubsequent to, a detection step.

Non-limiting examples of repetitive cycle ligation reactions include:ligase chain reaction (LCR, also known as oligonucleotide ligaseamplification (OLA)); gap LCR; ligation detection reaction (LDR);combined chain reaction (CCR which includes a combination of PCR andLCR); and methods coupled with polymerase chain reaction (PCR),including OLA-PCR, PCR-OLA and PCR-LDR.

In some embodiments, a ligation chain reaction (LCR) comprises a nucleicacid amplification method in which two pairs of oligonucleotide probeshybridize at adjacent positions to complementary strands of adouble-stranded target polynucleotide (Wu and Wallace 1989 Genomics4:560; Barany 1991 PNAS 88:189-193; U.S. Pat. No. 5,185,243 to Ullman,U.S. Pat. No. 5,427,930 to Birkenmeyer, U.S. Pat. No. 5,573,907 toCarrino U.S. Pat. No. 5,679,524 to Nikiforov, U.S. Pat. No. 5,869,252 toBouma, U.S. Pat. No. 6,858,412 to Willis; EP 0320308 to Backman, EP0336731 to Wallace, EP 0439182 to Backman; and WO 1990/01069 to Segav,WO 1989/12696 to Richards, WO 1989/09835 to Orgel and WO 1996/015271 toCarrino). The hybridized oligonucleotide probes can be ligated togetherwith ligase and aprataxin, then removed via denaturation. Multiplerounds of annealing, ligating and denaturing result in exponentialamplification of the target polynucleotide. In some embodiments, one orboth of the oligonucleotide probes can have substantially perfectcomplementarity or partial complementarity to the target polynucleotide.

In some embodiments, a modified LCR method comprises PCR reactions withlimited LCR (Wiedmann 1992 Appl Environ. Microbiol. 58:3443-3447;Wiedmann 1993 Appl Environ. Microbiol. 59:2743-2745).

In some embodiments, a gap LCR reaction comprises hybridizing a targetpolynucleotide to a pair of nucleic acid probe having 3′ overhang ends.The pair of probes can hybridize at adjacent positions on the templatepolynucleotide to form a gap of one to several bases (BackmanEP-A-0439182; Segev 1990 WO 90/01069; Birkenmeyer and Armstrong 1992 J.Clin. Microbiol. 30: 3089-3094; Abravaya 1995 NAR 23:675-682). The gapLCR reaction further comprises reacting the adjacent pair of probes witha DNA polymerase and nucleotides to fill in the gap, and covalentjoining with a ligase enzyme and aprataxin.

In some embodiments, a ligation detection reaction (LDR) comprises anucleic acid amplification method in which one pair of oligonucleotideprobes hybridize at adjacent positions to a target polynucleotide (Wuand Wallace 1989 Genomics 4:560; Barany 1991 PNAS 88:189-193; Wiedmann1992 Appl Environ. Microbiol. 58:3443-3447). The hybridizedoligonucleotide probes can be ligated together with ligase andaprataxin, then removed via denaturation. Multiple rounds of annealing,ligating and denaturing results in linear amplification of the targetpolynucleotide.

Non-limiting examples of specific applications of repetitive cycleligation reactions include: amplification of template, detection and/orquantification of the presence of a particular nucleic acid, e.g., in adiagnostic sample, ligation sequencing, single nucleotide polymorphism(SNP) analysis, SNP genotyping, mutation detection, identification ofsingle copy genes, detecting microsatellite repeat sequences, and DNAadduct mapping, among other things. See for example U.S. Pat. No.4,883,750 to Whitely, U.S. Pat. No. 4,988,617 to Landegren and Hood,U.S. Pat. No. 5,476,930 to Letsinger, U.S. Pat. No. 5,593,826 to Fung,U.S. Pat. No. 5,426,180 to Kool, U.S. Pat. No. 5,871,921 to Landegren;U.S. patent publication 2004/0110213 to Namsaraev; WO 1997/31256 toBarany, WO 2001/92579 to Wenz and Schroth; Xu and Kool 1999 Nucl. AcidsRes. 27: 875-881, Higgins 1979 Methods in Enzymology 68: 50-71, andEngler and Richardson 1982 In: “The Enzymes” Vol. 15, Boyer ed.,Academic Press, New York, N.Y., Landegren 1988 Science 241:1077-1080,Grossman 1994 Nucl. Acids. Res. 22:4527-4534, Bi and Stambrook 1997Nucl. Acids Res. 25:2949-51, and Zirvi 1999 Nucl. Acids Res. 27:e40

In some embodiments, a ligation reaction comprises joining together twonucleic acid ends via an enhanced ligation reaction. In someembodiments, an enhanced ligation reaction comprises joining together afirst end of a first nucleic acid with a first end of a second nucleicacid. In some embodiments, a first or second nucleic acid can be inunbound (e.g., non-immobilized or in solution) or can be attached to asurface (e.g., immobilized). For example, a second end of the firstand/or second nucleic acid can be attached to a surface.

In some embodiments, a 5′ or 3′ end can be attached to a surface. Insome embodiments, a nucleic acid end can be modified for attachment to asurface. For example, a 5′ or 3′ end can be modified to include an aminogroup that can bind to a carboxylic acid compound on a surface or aparticle. A 5′ end can include a phosphate group for reacting with anamine-coated surface (or particle) in the presence of a carbodiimide(e.g., water soluble carbodiimide). A nucleic acid can be biotinylatedat one end to bind with an avidin-like compound (e.g. streptavidin)attached to a surface.

In some embodiments, a surface can be planar, convex, concave, or anycombination thereof. A surface can be porous, semi-porous or non-porous.A surface can comprise an inorganic material, natural polymers,synthetic polymers, or non-polymeric material. A surface includes aflowcell, well, groove, channel, reservoir, filter, gel or inner wallsof a capillary. A surface can be coated with an acrylamide compound.

In some embodiments, a first and/or second nucleic acid can be attachedto a particle. In some embodiments, a particle comprises a shape that isspherical, hemispherical, cylindrical, barrel-shaped, toroidal,rod-like, disc-like, conical, triangular, cubical, polygonal, tubular,wire-like or irregular. In some embodiments, a particle comprises aniron core or a hydrogel or agarose (e.g., Sepharose™). A In someembodiments, a particle comprises paramagnetic material. In someembodiments, a particle comprises cavitation or pores orthree-dimensional scaffolds. In some embodiments, a particle comprises acoating of carboxylic acid compound or an amine compound for attachingnucleic acids. In some embodiments, a particle comprises a coating of anavidin-like compound (e.g., streptavidin) for binding biotinylatednucleic acids.

In some embodiments, a nucleic acid comprises a nucleic acid libraryconstruct. In some embodiments, one end of a nucleic acid libraryconstruct can be attached to a surface (planar surface, bead orparticle). In some embodiments, a nucleic acid library constructincludes any type of next generation sequencing library construct,including a fragment library (PCT/US11/24631), mate pair library(PCT/US11/54053), an RNA library (e.g., mRNA libraries, RNA-Seqlibraries, whole transcriptome libraries, cell-specific RNA libraries),chromatin immunoprecipitation (ChIP) library, and methylated DNAlibrary.

In some embodiments, beads or particles can be deposited to a surface.In some embodiments, beads or particles can be deposited to a surface ofa sequencing instrument. Sequencing reagents can be delivered to thedeposited beads or particles to conduct a sequencing reaction. In someembodiments, a sequencing reaction includes any next generationsequencing reaction, including: sequencing by oligonucleotide probeligation and detection (e.g., SOLiD™ from Life Technologies, WO2006/084131); probe-anchor ligation sequencing (e.g., Complete Genomics™or Polonator™); sequencing-by-synthesis (e.g., Genetic Analyzer, HiSeq™and MiSeq™ from Illumina); pyrophosphate sequencing (e.g., GenomeSequencer FLX from 454 Life Sciences); ion-sensitive sequencing (e.g.,Personal Genome Machine (PGM™) and Proton™ from Ion Torrent™ Systems,Life Technologies); and single molecule sequencing platforms (e.g.,HeliScope™ from Helicos).

In some embodiments, an immobilized nucleic acid library can besequenced by employing asequencing-by-oligonucleotide-probe-ligation-and-detection sequencingreaction (e.g., SOLiD sequencing reactions, WO 2006/084132, AppliedBiosystems which is now part of Life Technologies, Carlsbad, Calif.).

In some embodiments, one end of a nucleic acid library construct can beattached to a surface (e.g., Ion Sphere™ Particle, sold as a componentof the Ion Xpress Template Kit (Part No. 4469001). Immobilizing nucleicacid library constructs to Ion Sphere™ Particles can be performedessentially according to the protocols provided in the Ion Xpress™Template Kit v2.0 User Guide (Part No.: 4469004)). In some embodiments,a nucleic acid library construct includes a fragment library construct(PCT/US11/24631) or mate pair library construct (PCT/US11/54053). Insome embodiments, an immobilized nucleic acid library can be sequencedvia ion-sensitive or semiconductor sequencing methods, includingsequencing methods and platforms for an Ion Torrent PGM™ or Proton™sequencer (Life Technologies Corporation).

In some embodiments, an aprataxin enzyme comprises a polypeptide, or afragment thereof, that catalyzes nucleotide hydrolase activity.

In some embodiments, an aprataxin enzyme comprises a polypeptide, or afragment thereof, that catalyzes AMP hydrolase activity.

In some embodiments, an aprataxin enzyme comprises a polypeptide, or afragment thereof, that catalyzes nucleophilic release of an adenylategroup that is covalently linked to a terminal 5′-phosphate group.

In some embodiments, an aprataxin enzyme comprises a polypeptide, or afragment thereof, that catalyzes removal of an adenosine-phosphatemoiety that is covalently attached to a 5′ terminus of a nucleic acid.For example, an adenosine-phosphate moiety includes AMP, AMP-lysine,adenine monophosphoramidate and adenosine polyphosphates (Ahel 2006Nature 443:713-716; Takahashi 2007 Nucleic Acids Research 35:3797-3809;Kijas 2006 Journal Biol. Chem. 281:13939-13948; Rass 2007 Journal Biol.Chem. 282:9469-9474; Seidle 2005 Journal Biol. Chem. 280:20927-20931).

In some embodiments, an aprataxin enzyme can generate a terminal5′-phosphate group on a nucleic acid.

In some embodiments, an aprataxin enzyme can exhibit a proofreadingfunction during adenylate removal (Rass 2007 Journal Biol. Chem.282:9469-9474).

In some embodiments, an aprataxin enzyme comprises a polypeptide havingan amino acid sequence of a member of a histidine triad superfamily (orportion thereof) which include nucleotide binding proteins such asnucleotide hydrolases and transferases (Brenner 2002 Biochemistry41:9003-9014).

In some embodiments, an aprataxin enzyme can include at least onepolypeptide domain found in a naturally-occurring aprataxin enzyme,including: (i) a forkhead-associated domain (FHA) which interacts with aDNA repair/ligase complex such as ligases, polymerases, or asingle-strand break repair protein XRCC1 or XRCC4 (Harris 2009 HumanMol. Gen. 18:4102-4117); a histidine triad nucleotide hydrolase domain(HIT) which catalyzes adenylate hydrolase activity (Brenner 2002Biochemistry 41:9003-9014); and/or (iii) a zinc finger domain (ZF) whichinteracts with adenylated DNA to stabilize the DNA-enzyme complex (Rass2007 Journal Biol. Chem. 282:9469-9474).

In some embodiments, an aprataxin enzyme comprises a histidine triadmotif HxHxHxx, where x can be a hydrophobic amino acid. In someembodiments, a hydrophobic amino acid includes glycine, alanine, valine,leucine, isoleucine, methionine, phenylalanine, tryptophan and proline.For example, a histidine triad motif comprises an amino acid sequenceHis-Val-His-Leu-His-Val-Ile (in 3-letter amino acid code).

In some embodiments, an aprataxin enzyme comprises intact subunits,biologically-active fragments, mutant variants, truncated variants,recombinant variants, fusion variants, chimeric variants,naturally-occurring aprataxins, or non-naturally occurring aprataxins.Mutant variants include amino acid substitutions, insertions, and/ordeletions. In some embodiments, an aprataxin enzyme comprisesnaturally-occurring amino acids and/or amino acid analogs.

In some embodiments, an aprataxin enzyme can be isolated from a cell, orgenerated using recombinant DNA technology or chemical synthesismethods. In some embodiments, an aprataxin enzyme can be expressed inprokaryote, eukaryote, viral, or phage organisms. In some embodiments,an aprataxin enzyme can be post-translationally modified proteins orfragments thereof.

In some embodiments, an aprataxin enzyme can be a recombinant proteinwhich is produced by a suitable expression vector/host cell system. Anaprataxin can be encoded by a suitable recombinant expression vectorcarrying an inserted nucleotide sequence of an aprataxin enzyme, orportion thereof. An aprataxin nucleotide sequence can be operably linkedto a suitable expression vector. An aprataxin nucleotide sequence can beinserted in-frame into the suitable expression vector. A suitableexpression vector can replicate in a phage host, or a prokaryotic oreukaryotic host cell. A suitable expression vector can replicateautonomously in a host cell, or can be inserted into a host cell'sgenome and be replicated as part of a host genome. A suitable expressionvector can carry a selectable marker that confers resistance to drugs(e.g., kanamycin, ampicillin, tetracycline, chloramphenicol, or thelike) or a requirement for a nutrient. A suitable expression vector canhave one or more restriction sites for inserting a nucleic acid moleculeof interest. A suitable expression vector can include expression controlsequences for regulating transcription and/or translation of the encodedsequence. An expression control sequence can include: promoters (e.g.,inducible or constitutive), enhancers, transcription terminators, and/orsecretion signals. An expression vector can include a plasmid, cosmid,or phage vector. An expression vector can enter a host cell which canreplicate the vector, produce an RNA transcript of the insertedsequence, and/or produce protein encoded by the inserted sequence. Arecombinant aprataxin enzyme can include an affinity tag for enrichmentor purification, including a biotin, poly-His, GST and/or HA sequencetag. Methods for preparing suitable recombinant expression vectors andexpressing the RNA and/or protein encoded by the inserted sequences arewell known in the art (Sambrook et al, in: Molecular Cloning (1989)).

A polypeptide having aprataxin activity can include without limitationbacterial aprataxins, eukaryotic aprataxins, archaeal aprataxins, viralaprataxins and phage aprataxins. An aprataxin enzyme can becommercially-available. In some embodiments, an aprataxin enzymecomprises monkey (NCBI accession No. NP001253363), dog (NCBI accessionNo. NP001003355), rat (NCBI accession No. NP683687), cattle (NCBIaccession No. NP872595), pig (NCBI accession No. NP998899), zebrafish(NCBI accession No. NP999894), or frog aprataxin (NCBI accession No.NP001082689). In some embodiments, an aprataxin enzyme comprises humanaprataxin (FIG. 2A, SEQ ID NO:2). A polypeptide having aprataxinactivity can be encoded by a nucleic acid having a nucleotide sequenceas shown in FIG. 2B (SEQ ID NO:3). It will be appreciated by the skilledartisan that other nucleotide sequences that encode an aprataxinpolypeptide are possible given the degenerate genetic code.

In some embodiments, a ligase enzyme comprises a polypeptide, orfragment thereof, that catalyzes phosphodiester bond formation between aterminal 5′ phosphate end and a terminal 3′OH end to join together thetwo nucleic acid ends. In some embodiments, a ligase enzyme comprises aDNA ligase or RNA ligase.

In some embodiments, a ligase enzyme can catalyze closing asingle-stranded nick in a DNA duplex or joining together two ends of twonucleic acid strands or joining together two ends of a single nucleicacid strand (e.g., circularization).

In some embodiments, a ligase enzyme can catalyze joining togetherblunt-ends or overhang-ends.

In some embodiments, a ligase enzyme comprises a polypeptide (orfragment thereof) that exhibits ATP-dependent or NAD-dependent ligaseactivity.

In some embodiments, a ligase enzyme comprises a mesophilic orthermophilic ligase enzyme. In some embodiments, a thermostable ligaseenzyme can exhibit ligase activity at about 40-65° C., or about 65-85°C., or about 85-99° C. or higher temperature ranges.

In some embodiments, a ligase enzyme comprises a high ligation fidelityenzyme. For example, a ligase can discriminate the degree ofhybridization between opposing nucleic acid strands in a duplex.

In some embodiments, a ligase can have one or more of the followingactivities: (1) nucleophilic attack on ATP or NAD⁺ resulting in releaseof PPi or NMN and formation of a covalent ligase-adenylate intermediate;(2) transferring the adenylate to the 5′-end of a5′-phosphate-terminated DNA strand to form a DNA-adenylate complex(e.g., the 5′-phosphate oxygen of the DNA strand attacks the phosphorusof ligase-adenylate); and/or (3) formation of a covalent bond joiningthe polynucleotide termini and liberation of AMP (e.g., by the attack bythe 3′-OH on DNA-adenylate).

Optionally, a ligase can mediate any one or more of the following bondtransformations: from phosphoanhydride (ATP) to phosphoramidate(ligase-adenylate); from phosphoramidate (ligase-adenylate) tophosphoanhydride (DNA-adenylate); or from phosphoanhydride(DNA-adenylate) to phosphodiester (sealed DNA).

In some embodiments, a ligase enzyme comprises an E. coli DNA ligase,Taq DNA ligase, 9° N DNA ligase, or T4 DNA ligase.

In some embodiment, a ligase enzyme comprises a small footprint ligaseenzyme which can ligate short nucleic acids.

In some embodiments, a small footprint ligase can join together apolynucleotide to an oligonucleotide that is 8, 7, 6, 5, 4, 3 or 2nucleotides in length. Ligation of such oligonucleotides can be tooligonucleotides of the same length or of different length or to apolynucleotide. For example, an oligonucleotide of 2 or 3 nucleotides inlength can be ligated to an oligonucleotide of 2, 3, 4, 5, 6, 7, 8 ormore nucleotides in length or to longer oligonucleotides or to apolynucleotide.

Exemplary ligases can comprise a polypeptide sequence that is homologousto or a variant of a known ligase or small footprint ligase sequence orany portion thereof. Exemplary ligases and small footprint ligasesoptionally have amino acid sequence identity of at least 70%, optionallyat least 85%, optionally at least 90, 95%, 97% or 99%, with a knownligase or known small footprint ligase.

Representative examples of small footprint ligases include a Hin DNAligases (e.g., DLX, DLXd, and DLXd2 ligases), a Chlorella Virus ligase(FIG. 3, SEQ ID NO:3), and MnM ligase (FIG. 4, SEQ ID NO:4).

In some embodiments, a small footprint ligase can be derived from a HinDNA ligase (e.g., DLX, DLXd or DLXd2) (FIGS. 5-8, SEQ ID NOS:5-8,respectively) or any fragment or variant thereof that still retains oneor more mutant residues shown in any of FIGS. 3-8 (SEQ ID NOS:3-8,respectively), and/or has one or more C-terminal amino acids deleted,e.g., 22 C-terminal amino acids deleted. For example, a mutant Hin DNAligase can be at least 70% identical to Hin D ligase sequence providedin any of FIGS. 5-8 (SEQ ID NOS:5-8, respectively) or in GenBankAccession No. P44121, which ligase comprises an amino acid mutation atposition 193 of the Hin D ligase sequence provided in FIGS. 5-8 (SEQ IDNOS:5-8, respectively) or in GenBank Accession No. P44121. Optionallythe amino acid mutation consists of changing the glycine at position 193to aspartic acid or glutamic acid.

In some embodiments, a small footprint ligase can be derived from aChlorella virus (ChVLig) (Ho 1997 J Virol, 71(3):1931-19374) or aParamecium Bursaria Chlorella Virus ligase (PBCV ligase) (Odell andShuman 1999 Journal of Biol. Chem. 274:14032-14039) or a functionalfragment or variant thereof. For example a small footprint ligase cancomprise any one or more domains characteristic of a ligase, e.g., anN-terminal nucleotidyltransferase (NTase) domain and/or a C-terminaloligonucleotide binding domain (OB domain). In some embodiments, anoligonucleotide binding domain optionally comprises a five-strandedantiparallel beta-barrel plus an alpha-helix. In some embodiments, theN-terminal nucleotidyltransferase domain includes an adenylate-bindingpocket composed of the six peptide motifs that define the covalent NTaseenzyme family of polynucleotide ligases.

Optionally, the N-terminal nucleotidyltransferase domain can compriseany one or more of the ligase amino acid motifs I, Ia, III, IIIa, IV, V,and VI. For example, Motif I (e.g., K×DG×R or a “KXDG” motif) optionallycomprises a lysine. Exemplary sequences for each motif in a Chlorellavirus ligase comprise ATPKIDGIR (motif I), SRT (motif Ia), EGSDGEIS(motif III), YWFDY (motif IIIa), EGVMIR (motif IV), LLKMK (motif V).

In some embodiments, a Motif 1 comprises a lysine residue. Otherexamples of motif I include CELKLDGLA, VEHKVDGLS, CEPKLDGLA, CELKLDGVA,AEIKYDGVR, CEYKYDGQR, VDYKYDGER, FEIKYDGAR, FEGKWDGYR, AREKIHGTN,ACEKVHGTN, ILTKEDGSL, and VEEKVDGYN.

Examples of motif Ia include TRG, SRT, SRR, SRN, SRS, KRT, KRS, SKG andTRG.

Examples of motif III include LEVRGEVF, VEVRGECY, LEVRGEVY, LEARGEAF,FMLDGELM, EGSDGEIS, FILDTEAV, FIIEGEIV, AIVEGELV, VVLDGEAV, YQVFGEFA,LVLNGELF, FTANFEFV and LILVGEMA.

Examples of motif IIIa include FCYGV, FLYTV, TFYAL, ICHGL, NAYGI, FVYGL,KLYAI, YWFDY, YAFDI, FLFDL, NLFDV, WAFDL, YVFDI, FAFDI, ILLNA, andFLFDV.

Examples of motif IV include DGVVIK, DGIVIK, DGVVVK, DGTVLK, EGLIVK,EGVMIR, EGLMVK, EGVMVK, EGLMAK, EGVIAK, EGYVLK, EGVVIR, EGYVAV, andEGIIMK.

Examples of motif V include AVAFK, AIAYK, ALAYK, AIAYK,WWKMK, LLKMK,WLKLK, WIKLK, WLKIK, WVKDK, AIKCK, IIKLR, HFKIK and IVKYV.

In some embodiments, small footprint ligases include Paramecium bursariaChlorella virus (PBCV) ligase enzyme. In some embodiments, the PBCVligase enzyme comprises an amino acid sequence shown in FIG. 3 (SEQ IDNO:3). In some embodiments, the aprataxin enzyme comprises an amino acidsequence shown in FIG. 2A. In some embodiments, the PBCV ligase enzymeexhibits activity of a small footprint ligase enzyme. In someembodiments, the nick is contacted with about 0.001-2 mg/ml of PBCVligase enzyme and about 0.001-2 mg/ml of aprataxin enzyme. In someembodiments, the nick is contacted with about 0.01-1 mg/ml of PBCVligase enzyme and about 0.01-1 mg/ml of aprataxin enzyme. In someembodiments, the PBVC ligase and aprataxin can be stored in a solutioncomprising about 1-25 mM Tris (pH about 7.5), about 0.001-0.5 EDTA,about 10-200 mM KCl, about 0.001-2 mM DTT, about 0.001-0.5% Tween 20,and about 10-75% glycerol. In some embodiments, a SOLiD sequencingreaction can be conducted with nucleic acid-templated beads, labeledoligonucleotide probes (that can hybridize to the nucleic acid templateto form nicks), PBCV ligase enzyme, and aprataxin enzyme underconditions suitable for ligation reactions.

In some embodiments, small footprint ligases include those described inU.S. Ser. Nos. 61/433,488 filed on Jan. 17, 2011, U.S. 61/433,502, filedon Jan. 17, 2011, U.S. 61/474,168, filed on Apr. 11, 2011, U.S.61/474,205, filed on Apr. 11, 2011, and published PCT application No.PCT/US2012/021465, filed on Jan. 17, 2012, all these references arehereby incorporated by reference in their entireties.

In some embodiments, a small footprint ligase comprises an RNA-cappingenzyme, or does not comprise an RNA-capping enzyme.

In some embodiments, methods for enhancing a nucleic acid ligationreaction comprise joining together nucleic acid ends (ends of nucleicacids, oligonucleotides or polynucleotides). In some embodiments,nucleic acids comprise single-stranded or double-stranded nucleic acids.In some embodiments, nucleic acids comprise DNA, RNA or chimericRNA/DNA. In some embodiments, nucleic acids comprise isolated nucleicacids in any form including chromosomal, genomic, organellar (e.g.,mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned,subcloned, amplified (e.g., PCR amplified), cDNA, RNA such as precursormRNA or mRNA, oligonucleotide, or any type of nucleic acid library. Insome embodiments, nucleic acids can be isolated from any sourceincluding from organisms such as prokaryotes, eukaryotes (e.g., humans,plants and animals), fungus, and viruses; cells; tissues; normal ordiseased cells or tissues; organs; body fluids; environmental samples;culture samples; or synthesized nucleic acid molecules prepared usingrecombinant molecular biology or chemical synthesis methods. In someembodiments, nucleic acids can be chemically synthesized to include anytype of natural and/or analog nucleic acid. In some embodiments, nucleicacids can be isolated from a formalin-fixed tissue, or from aparaffin-embedded tissue, or from a formalin-fix paraffin-embedded(FFPE) tissue. In some embodiments, amplified nucleic acids can begenerated by one or more cycles of a template walking reactioncomprising primer extension of a primer hybridized to a template strandto produce an extended strand, and partial or incomplete denaturation ofthe extended strand from the template stand (U.S. non-provisionalapplication Ser. No. 13/328,844, filed on Dec. 16, 2011).

In some embodiments, nucleic acids joined together with ligase andaprataxin can be sequenced using methods that detect one or morebyproducts of nucleotide incorporation. The detection of polymeraseextension by detecting physicochemical byproducts of the extensionreaction, can include pyrophosphate, hydrogen ion, charge transfer,heat, and the like, as disclosed, for example, in Pourmand et al, Proc.Natl. Acad. Sci., 103: 6466-6470 (2006); Purushothaman et al., IEEEISCAS, IV-169-172; Rothberg et al, U.S. Patent Publication No.2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); Sakata et al., Angew. Chem. 118:2283-2286 (2006); Esfandyapouret al., U.S. Patent Publication No. 2008/01666727; and Sakurai et al.,Anal. Chem. 64: 1996-1997 (1992).

Reactions involving the generation and detection of ions are widelyperformed. The use of direct ion detection methods to monitor theprogress of such reactions can simplify many current biological assays.For example, template-dependent nucleic acid synthesis by a polymerasecan be monitored by detecting hydrogen ions that are generated asnatural byproducts of nucleotide incorporations catalyzed by thepolymerase. Ion-sensitive sequencing (also referred to as “pH-based” or“ion-based” nucleic acid sequencing) exploits the direct detection ofionic byproducts, such as hydrogen ions, that are produced as abyproduct of nucleotide incorporation. In one exemplary system forion-based sequencing, the nucleic acid to be sequenced can be capturedin a microwell, and nucleotides can be floated across the well, one at atime, under nucleotide incorporation conditions. The polymeraseincorporates the appropriate nucleotide into the growing strand, and thehydrogen ion that is released can change the pH in the solution, whichcan be detected by an ion sensor. This technique does not requirelabeling of the nucleotides or expensive optical components, and allowsfor far more rapid completion of sequencing runs. Examples of suchion-based nucleic acid sequencing methods and platforms include the IonTorrent PGM™ or Proton™ sequencer (Life Technologies Corporation).

In some embodiments, one or more nucleic acid fragments produced usingthe methods, systems and kits of the present teachings can be used as asubstrate for a biological or chemical reaction that is detected and/ormonitored by a sensor including a field-effect transistor (FET). Invarious embodiments the FET is a chemFET or an ISFET. A “chemFET” orchemical field-effect transistor, is a type of field effect transistorthat acts as a chemical sensor. It is the structural analog of a MOSFETtransistor, where the charge on the gate electrode is applied by achemical process. An “ISFET” or ion-sensitive field-effect transistor,is used for measuring ion concentrations in solution; when the ionconcentration (such as H+) changes, the current through the transistorwill change accordingly. A detailed theory of operation of an ISFET isgiven in “Thirty years of ISFETOLOGY: what happened in the past 30 yearsand what may happen in the next 30 years,” P. Bergveld, Sens. Actuators,88 (2003), pp. 1-20.

In some embodiments, the FET may be a FET array. As used herein, an“array” is a planar arrangement of elements such as sensors or wells.The array may be one or two dimensional. A one dimensional array can bean array having one column (or row) of elements in the first dimensionand a plurality of columns (or rows) in the second dimension. The numberof columns (or rows) in the first and second dimensions may or may notbe the same. The FET or array can comprise 102, 103, 104, 105, 106, 107or more FETs.

In some embodiments, one or more microfluidic structures can befabricated above the FET sensor array to provide for containment and/orconfinement of a biological or chemical reaction. For example, in oneimplementation, the microfluidic structure(s) can be configured as oneor more wells (or microwells, or reaction chambers, or reaction wells,as the terms are used interchangeably herein) disposed above one or moresensors of the array, such that the one or more sensors over which agiven well is disposed detect and measure analyte presence, level,and/or concentration in the given well. In some embodiments, there canbe a 1:1 correspondence of FET sensors and reaction wells.

Microwells or reaction chambers are typically hollows or wells havingwell-defined shapes and volumes which can be manufactured into asubstrate and can be fabricated using conventional microfabricationtechniques, e.g. as disclosed in the following references: Doering andNishi, Editors, Handbook of Semiconductor Manufacturing Technology,Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMSand Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al,Silicon Micromachining (Cambridge University Press, 2004); and the like.Examples of configurations (e.g. spacing, shape and volumes) ofmicrowells or reaction chambers are disclosed in Rothberg et al, U.S.patent publication 2009/0127589; Rothberg et al, U.K. patent applicationGB24611127.

In some embodiments, the biological or chemical reaction can beperformed in a solution or a reaction chamber that is in contact with orcapacitively coupled to a FET such as a chemFET or an ISFET. The FET (orchemFET or ISFET) and/or reaction chamber can be an array of FETs orreaction chambers, respectively.

In some embodiments, a biological or chemical reaction can be carriedout in a two-dimensional array of reaction chambers, wherein eachreaction chamber can be coupled to a FET, and each reaction chamber isno greater than 10 μm³ (i.e., 1 pL) in volume. In some embodiments eachreaction chamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pLin volume. A reaction chamber can optionally be 22, 32, 42, 52, 62, 72,82, 92, or 102 square microns in cross-sectional area at the top.Preferably, the array has at least 102, 103, 104, 105, 106, 107, 108,109, or more reaction chambers. In some embodiments, the reactionchambers can be capacitively coupled to the FETs.

FET arrays as used in various embodiments according to the disclosurecan be fabricated according to conventional CMOS fabricationstechniques, as well as modified CMOS fabrication techniques and othersemiconductor fabrication techniques beyond those conventionallyemployed in CMOS fabrication. Additionally, various lithographytechniques can be employed as part of an array fabrication process.

Exemplary FET arrays suitable for use in the disclosed methods, as wellas microwells and attendant fluidics, and methods for manufacturingthem, are disclosed, for example, in U.S. Patent Publication No.20100301398; U.S. Patent Publication No. 20100300895; U.S. PatentPublication No. 20100300559; U.S. Patent Publication No. 20100197507,U.S. Patent Publication No. 20100137143; U.S. Patent Publication No.20090127589; and U.S. Patent Publication No. 20090026082, which areincorporated by reference in their entireties. Examples of an array caninclude Ion Torrent™ System arrays, such as the 314™, 316™ and 318™Chips (Life Technologies) used in conjunction with an Ion Torrent™ PGMor Proton™ Sequencer (Life Technologies, Part No. 4462917).

In one aspect, the disclosed methods, compositions, systems, apparatusesand kits can be used for carrying out label-free nucleic acidsequencing, and in particular, ion-based nucleic acid sequencing. Theconcept of label-free detection of nucleotide incorporation has beendescribed in the literature, including the following references that areincorporated by reference: Rothberg et al, U.S. patent publication2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470(2006). Briefly, in nucleic acid sequencing applications, nucleotideincorporations are determined by measuring natural byproducts ofpolymerase-catalyzed extension reactions, including hydrogen ions,polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase).

In some embodiments, the disclosure relates generally to methods forjoining together nucleic acids with ligase and aprataxin to generate anucleic acid construct that can be sequenced using hydrogenion-sensitive sequencing methods.

In some embodiments, the nucleic acid construct can be sequenced usingan ion-sensitive sequencing method. In some embodiments, the sequencingmethod is performed by incorporating one or more nucleotides in atemplate-dependent fashion into a newly synthesized nucleic acid strand.

Optionally, the methods can further include producing one or more ionicbyproducts of such nucleotide incorporation.

In some embodiments, the methods can further include detecting theincorporation of the one or more nucleotides into the sequencing primer.Optionally, the detecting can include detecting the release of hydrogenions.

In another embodiment, the disclosure relates generally to a method forsequencing a nucleic acid, comprising: (a) producing a nucleic acidconstruct according to the methods disclosed herein; (b) disposing aplurality of nucleic acid constructs into a plurality of reactionchambers, wherein one or more of the reaction chambers are in contactwith a field effect transistor (FET). Optionally, the method furtherincludes contacting at least one of the nucleic acid constructs disposedinto one of the reaction chambers with a polymerase, therebysynthesizing a new nucleic acid strand by sequentially incorporating oneor more nucleotides into a nucleic acid molecule. Optionally, the methodfurther includes generating one or more hydrogen ions as a byproduct ofsuch nucleotide incorporation. Optionally, the method further includesdetecting the incorporation of the one or more nucleotides by detectingthe generation of the one or more hydrogen ions using the FET.

In some embodiments, the detecting includes detecting a change involtage and/or current at the at least one FET within the array inresponse to the generation of the one or more hydrogen ions.

In some embodiments, the FET can be selected from the group consistingof: ion-sensitive FET (isFET) and chemically-sensitive FET (chemFET).

One exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™ orProton™ sequencer (Life Technologies), which is an ion-based sequencingsystem that sequences nucleic acid templates by detecting hydrogen ionsproduced as a byproduct of nucleotide incorporation. Typically, hydrogenions are released as byproducts of nucleotide incorporations occurringduring template-dependent nucleic acid synthesis by a polymerase. TheIon Torrent PGM™ or Proton™ sequencer detects the nucleotideincorporations by detecting the hydrogen ion byproducts of thenucleotide incorporations. The Ion Torrent PGM™ or Proton™ sequencer caninclude a plurality of nucleic acid templates to be sequenced, eachtemplate disposed within a respective sequencing reaction well in anarray. The wells of the array can each be coupled to at least one ionsensor that can detect the release of H⁺ ions or changes in solution pHproduced as a byproduct of nucleotide incorporation. The ion sensorcomprises a field effect transistor (FET) coupled to an ion-sensitivedetection layer that can sense the presence of H⁺ ions or changes insolution pH. The ion sensor can provide output signals indicative ofnucleotide incorporation which can be represented as voltage changeswhose magnitude correlates with the H⁺ ion concentration in a respectivewell or reaction chamber. Different nucleotide types can be flowedserially into the reaction chamber, and can be incorporated by thepolymerase into an extending primer (or polymerization site) in an orderdetermined by the sequence of the template. Each nucleotideincorporation can be accompanied by the release of H⁺ ions in thereaction well, along with a concomitant change in the localized pH. Therelease of H⁺ ions can be registered by the FET of the sensor, whichproduces signals indicating the occurrence of the nucleotideincorporation. Nucleotides that are not incorporated during a particularnucleotide flow may not produce signals. The amplitude of the signalsfrom the FET can also be correlated with the number of nucleotides of aparticular type incorporated into the extending nucleic acid moleculethereby permitting homopolymer regions to be resolved. Thus, during arun of the sequencer multiple nucleotide flows into the reaction chamberalong with incorporation monitoring across a multiplicity of wells orreaction chambers can permit the instrument to resolve the sequence ofmany nucleic acid templates simultaneously. Further details regardingthe compositions, design and operation of the Ion Torrent PGM™ orProton™ sequencer can be found, for example, in U.S. patent applicationSer. No. 12/002,781, now published as U.S. Patent Publication No.2009/0026082; U.S. patent application Ser. No. 12/474,897, now publishedas U.S. Patent Publication No. 2010/0137143; and U.S. patent applicationSer. No. 12/492,844, now published as U.S. Patent Publication No.2010/0282617, all of which applications are incorporated by referenceherein in their entireties.

In some embodiments, the disclosure relates generally to use of nucleicacid constructs produced using any of the methods, systems and kits ofthe present disclosure in methods of ion-based sequencing. Use of suchnucleic acid constructs in ion-based sequencing reactions can beadvantageous because the methods of the disclosure permit isolation ofpolynucleotides (e.g., tags) of a desired size that can be selected tomatch the read length capacity of the ion-based sequencing system.

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations can be detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzedextension reactions. In one embodiment, templates each having a primerand polymerase operably bound can be loaded into reaction chambers (suchas the microwells disclosed in Rothberg et al, cited herein), afterwhich repeated cycles of nucleotide addition and washing can be carriedout. In some embodiments, such templates can be attached as clonalpopulations to a solid support, such as a particle, bead, or the like,and said clonal populations are loaded into reaction chambers. As usedherein, “operably bound” means that a primer is annealed to a templateso that the primer's 3′ end may be extended by a polymerase and that apolymerase is bound to such primer-template duplex, or in closeproximity thereof so that binding and/or extension takes place whenevernucleotides are added.

In each addition step of the cycle, the polymerase can extend the primerby incorporating added nucleotide only if the next base in the templateis the complement of the added nucleotide. If there is one complementarybase, there is one incorporation, if two, there are two incorporations,if three, there are three incorporations, and so on. With each suchincorporation there is a hydrogen ion released, and collectively apopulation of templates releasing hydrogen ions changes the local pH ofthe reaction chamber. The production of hydrogen ions is monotonicallyrelated to the number of contiguous complementary bases in the template(as well as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereare a number of contiguous identical complementary bases in the template(i.e. a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, can be proportional tothe number of contiguous identical complementary bases. If the next basein the template is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, an additional stepcan be performed, in which an unbuffered wash solution at apredetermined pH is used to remove the nucleotide of the previous stepin order to prevent misincorporations in later cycles. In someembodiments, the after each step of adding a nucleotide, an additionalstep can be performed wherein the reaction chambers are treated with anucleotide-destroying agent, such as apyrase, to eliminate any residualnucleotides remaining in the chamber, which may result in spuriousextensions in subsequent cycles.

In one exemplary embodiment, different kinds of nucleotides are addedsequentially to the reaction chambers, so that each reaction can beexposed to the different nucleotides one at a time. For example,nucleotides can be added in the following sequence: dATP, dCTP, dGTP,dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposure followed bya wash step. The cycles may be repeated for 50 times, 100 times, 200times, 300 times, 400 times, 500 times, 750 times, or more, depending onthe length of sequence information desired.

In some embodiments, sequencing can be performed with an Ion Torrent™PGM™ or Proton™ sequencer. For example, nucleic acid constructs preparedas disclosed herein can be clonally amplified on Ion Sphere™ Particlesas part of the Ion Xpress™ Template Kit (Life Technologies Part No.4469001). Template preparation can be performed essentially accordinglyto the protocols provided in the Ion Xpress™ Template Kit v2.0 UserGuide (Life Technologies, Part No. 4469004). The amplified DNA can thenbe sequenced on the Ion PGM™ sequencer (Ion Torrent™, Life Technologies,Part No. 4462917) essentially according to the protocols provided in theIon Sequencing Kit v2.0 User Guide (Ion Torrent™, Life Technologies,Part No. 4469714) and using the reagents provided in the Ion SequencingKit (Ion Torrent™, Life Technologies, Part No. 4468997) and the Ion 314™Chip Kit (Ion Torrent™, Life Technologies, Part No. 4462923).

In some embodiments, systems comprise at least one enzyme for DNAadenylation removal and at least one enzyme for nucleic acid ligation.In some embodiments, systems comprise at least one aprataxin enzyme andat least one ligase enzyme. In some embodiments, systems can include anycombination of nucleic acid ends, at least on aprataxin enzyme and/or atleast one ligase enzyme. In some embodiments, systems can furtherinclude any combination of components that may be required for aprataxinor ligase enzymatic activity, including buffers, reagents, ATP, NADand/or ions (e.g., magnesium, manganese, cobalt, or calcium).

In some embodiments, kits comprise any combination of: buffers,reagents, ATP, NAD, ions (e.g., KCl, K-acetate, NH₄-acetate,K-glutamate, NH₄Cl, or ammonium sulfate) or cations (e.g., magnesium,manganese, cobalt, or calcium), at least one aprataxin enzyme, and/or atleast one ligase enzyme. In some embodiments, a buffer can include Tris,Tricine, HEPES, MOPS, ACES, MES, or inorganic buffers such as phosphateor acetate-based buffers which can provide a pH range of about 4-12. Insome embodiments, a buffer can include chelating agents such as EDTA orEGTA. In some embodiments, a buffer can include dithiothreitol (DTT),glycerol, spermidine, BSA (bovine serum albumin) and/or Tween. Kits canfurther comprise test nucleic acids for conducting control reactions forenhanced ligation. In some embodiments, a kit can omit any of thesecomponents. For example, a kit can include at least one aprataxin enzymeor at least one ligase enzyme. In some embodiments, kits can include anycombination of: various enzymes to conduct reactions such as nucleicacid ligation and/or DNA adenylation removal. In some embodiments, thekits can include a set of instructions and genome assembly guides can beincluded. Such material can be, for example, in print or in digitalform.

What is claimed:
 1. A method for sequencing nucleic acids, comprising:a) forming a plurality of polynucleotide duplexes each having a nick, by(i) hybridizing a first template polynucleotide with a first and secondprobe oligonucleotide so that the first and second probeoligonucleotides abut each other to form a first nick, wherein thesecond probe oligonucleotide is labeled with a first distinctivereporter moiety, and wherein the 5′ end of the first templatepolynucleotide is attached to a support, and (ii) hybridizing a secondtemplate polynucleotide with a third and fourth probe oligonucleotide sothat the third and fourth oligonucleotides abut each other to form asecond nick wherein, the fourth probe oligonucleotide is labeled with asecond distinctive reporter moiety, wherein the first and secondtemplate polynucleotides include different sequences, wherein the secondand fourth probe oligonucleotides include different sequences, whereinthe first and second distinctive reporter moieties distinguish thedifferent sequences of the second and fourth probe oligonucleotidesrespectively, and wherein the 5′ end of the second templatepolynucleotide is attached to the support; b) closing the first andsecond nick to form a first and second ligation product respectively byconducting a nucleic acid ligation reaction with a ligase and anaprataxin enzyme that catalyzes removal of an adenylate group from aterminal 5′ phosphate of a nucleic acid; and c) detecting the firstdistinctive reporter moiety and detecting the second distinctivereporter moiety; d) forming a plurality of polynucleotide duplexes eachhaving a nick, by (i) hybridizing the first template polynucleotide witha fifth probe oligonucleotide so that the first ligation product and thefifth probe oligonucleotide abut each other to form a third nick,wherein the fifth probe oligonucleotide is labeled with a thirddistinctive reporter moiety, and (ii) hybridizing the second templatepolynucleotide with a sixth probe oligonucleotide so that the secondligation product and the sixth probe oligonucleotide abut each other toform a fourth nick, wherein the sixth probe oligonucleotide is labeledwith a fourth distinctive reporter moiety, wherein the third and fourthreporter moieties distinguish the different sequences of the fifth andsixth probe oligonucleotides respectively; e) closing the third andfourth nick to form a third and fourth ligation product respectively, byconducting a nucleic acid ligation reaction with a ligase and anaprataxin enzyme that catalyzes removal of an adenylate group from aterminal 5′ phosphate of a nucleic acid; f) detecting the thirddistinctive reporter moiety thereby detecting the sequence of a portionof the first template polynucleotide that hybridizes to the fiftholigonucleotide, and detecting the fourth distinctive reporter moietythereby detecting the sequence of a portion of the second templatepolynucleotide that hybridizes to the sixth oligonucleotide, wherein thefirst, second, third and fourth distinctive reporter moieties comprisedifferent fluorophores; and g) denaturing the third and fourth ligationproducts from the first and second polynucleotides respectively andrepeating steps a) through f) with additional probe oligonucleotideslabeled with distinctive reporter moieties, optionally repeating formultiple repeated cycles; thereby detecting the sequence of a firsttemplate polynucleotide and detecting the sequence of a second templatepolynucleotide.
 2. The method of claim 1, wherein the support comprisesa planar support.
 3. The method of claim 1, wherein the planar supportcomprises a plurality of wells arranged in an array.
 4. The method ofclaim 1, wherein the planar support comprises 100-10⁷ wells.
 5. Themethod of claim 4, wherein each well is capacitively coupled to a fieldeffect transistor (FET).
 6. The method of claim 5, wherein the FETdetects protons.
 7. The method of claim 1, wherein the support comprisesa bead.
 8. The method of claim 7, further comprising depositing the beadonto an array.
 9. The method of claim 8, wherein the array comprises aplurality of wells.
 10. The method of claim 8, wherein the arraycomprises 100-10⁷ wells.
 11. The method of claim 8, wherein each well iscapacitively coupled to a field effect transistor (FET).
 12. The methodof claim 11, wherein the FET detects pyrophosphate, hydrogen ions,charge transfer or heat.
 13. The method of claim 1, wherein theaprataxin enzyme comprise the amino acid sequence according to SEQ IDNO:1.
 14. The method of claim 1, wherein the ligase comprises a smallfootprint ligase comprising the amino acid sequence according to any oneof SEQ ID NOS: 6-8.
 15. The method of claim 1, wherein the first orsecond oligonucleotide includes an internal or terminal scissile linkagewhich is selected from a group consisting of a phosphoramidate,phosphorothioate, or phosphorodithiolate linkage.
 16. The method ofclaim 1, wherein the first and second distinctive report moietiescomprise different fluorophores.
 17. The method of claim 1, furthercomprising: denaturing the first and second ligation product from thefirst and second polynucleotides respectively.